Abstract: Cancer immunotherapy has taken center stage in the fight against cancer. The mAb and their related counterparts remain at the forefront of cancer immunotherapy applications. However, new and improved therapeutics and/or treatment methods that may overcome their production, administration, and pharmacological limitations, including the evolution of treatment resistance are still in widespread demand. Key to the development of technological innovations that may overcome these limitations are small to intermediate size molecules with the targeting and effector functions of antibodies. Towards this goal, our research objective is to mimic antibody targeting and effector functions with semi-synthetic peptide-protein bioconjugates. In this application, the peptide sequence, Pep42, has been selected to target the Glucose Regulated Protein of 78 kilodalton (GRP78) on the surface of tumors but not on healthy tissues. The selected tumor antigen, B7-H6, exhibits NKp30 receptor binding on NK cells and immunostimulatory activity by the release of inflammatory cytokines that ultimately trigger tumor lysis and death. However, the downregulation or shedding of B7-H6 from tumors reduces NKp30 activation of NK cells, ultimately diminishing their anti-tumor immune responses. Therefore, we propose the development of new Pep42-B7-H6 conjugates that provide effective NK cell targeting and killing of GRP78 overexpressing tumors that lack cell surface B7-H6. The proposed project will be addressed by two specific aims: 1) the preparation of the Pep42-B7-H6 conjugates and 2) evaluation of their anti-cancer effects. Significantly, the Pep42-B7-H6 conjugates are anticipated to potentiate cancer immunotherapy of resilient tumors that evade NK cell-dependent immunity while providing insights into the production of semi-synthetic antibody mimics.